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. Author manuscript; available in PMC: 2016 May 1.
Published in final edited form as: Fungal Genet Biol. 2014 Oct 13;78:49–54. doi: 10.1016/j.fgb.2014.10.003

Cryptococcosis Diagnosis and Treatment: What Do We Know Now

John R Perfect 1, Tihana Bicanic 2
PMCID: PMC4395512  NIHMSID: NIHMS638590  PMID: 25312862

From its humble beginnings as a single case report in 1895 to its worldwide explosion of disease with a million cases per year as the HIV epidemic peaked (Park et al., 2009), Cryptococcus has achieved a major place in clinical mycology as the “sugar-coated” (polysaccharide capsule) yeast that can efficiently kill the susceptible host with and without treatment. Furthermore, it represents a major fungal model system for the study and development of principles in fungal pathogenesis, diagnosis, and treatment. This yeast has a mature molecular biology foundation with over 100 genetic loci linked to its virulence composite and widespread genomic, transcriptomic and proteomic studies available for study. Its diagnosis is supported on several levels by routine culture techniques, a well-described histopathology and the best validated serological tests in all of medical mycology. Finally, its treatment principles are supported by some of the most robust clinical studies in invasive fungal diseases.

The Dynamic Yeast

Cryptococcosis is caused primarily by two fungal species, Cryptococcus neoformans and Cryptococcus gattii, and these species have been further sub-divided into several genotypes (VNI, VII, VNB, VNIV, and VGI-IV) and only in rare circumstances are other cryptococcal species found to cause human disease. The taxonomy and population genetics of these two species continue to be evaluated and with the widespread and effective use of Multiple Locus Sequence Typing (MLST) analysis and whole genome sequencing, understanding strain heritage has become even more precise. Of the human pathogenic fungi, the two Cryptococcus species represent one of the most robust MLST fungal databases and with whole genome sequencing platforms, detailed analyses of strains will greatly expand soon. Recent genomic studies have begun to emphasize a very important principle: the cryptococcal genomes display impressive plasticity and capacity for microevolution. These changes in the cryptococcal genome structure can occur with strain passage in vitro or be frequently observed in the stress environment of the human subrachnoid space (Hu et al., 2011; Janbon et al., 2014). Furthermore, it has been observed that even within clinical strains isolated from infected patients that there are genomic differences noted and certain specific genotypes have been associated with a poor clinical prognosis (Wiesner et al., 2012). These observations challenge medical mycologists to characterize the precise whole genetic virulence composite of these strains beyond their obvious genetic controls of the classical virulence phenotypes such as capsule and melanin production, high temperature growth, and phospholipase/urease activity.

The virulence composite of this encapsulated yeast is clearly multigenetic, changing, and complex. First, it has been shown that different strains of Cryptococcus spp. at the site of infection, such as in the human subarachnoid space, functionally react both similarly and differently with respect to their regulation of stress responses and transporter genes in order to survive in this harsh host environment (Chen et al., 2014). Second, the dynamic mechanism of heteroresistance to fluconazole in Cryptococcus spp. that has been elegantly described (Sionov et al., 2010) has now importantly been shown to be relevant in vivo (Sionov et al., 2013). For instance, drug resistance can result from stress-induced chromosomal plasticity and aneuploidy formation involving gene duplications of the target(s) or azole efflux pumps. Therefore, under in vivo stress there is resistance to high concentrations of fluconazole through gene duplications for the ergosterol targets or efflux pumps. This drug resistant phenotypic observation likely occurs in patients with a high burden of yeasts in cerebrospinal fluid (CSF) during the fungistatic effects of fluconazole treatment. However, when the yeast is isolated from the host and grown in vitro under fully nutritious conditions, this aneuploidy is resolved and the yeast is now again phenotypically susceptible to the azole. This disconnect between in vitro antifungal susceptibility testing for Cryptococcus may be fundamentally confusing to the clinician simply due to the genetics and pathobiology of the yeast. It potentially adds to the uncertainty of precise “clinical in vitro break-points” for azoles in cryptococcosis management. Third, even the simple but dynamic human biological fluid, CSF, represents a dynamic challenge to this yeast which must meet this harsh environment with a series of genetic controls for adaptation (Lee et al., 2012). Fourth, it is also important to emphasize that in many respects the general phenotypic and genetic responses are used similarly by both C. neoformans and C. gattii strains. However, there are some differences between these species, reflecting their approximately 40 million year genetic separation and thus pathophysiology will not be exactly the same (Ngamskulrungroj et al., 2012; Ngamskulrungroj et al., 2009).

Although we are beginning to understand the population genetics of this clinically important group of basidiomycetes and have been able to identify genetic variations which will be helpful for epidemiological purposes and even possibly detect differences in relapse vs persistent cryptococcal strains (Van Wyk et al., 2014), does the clinician yet benefit from this genetic scrutiny and is this information ready for the clinical microbiology laboratory? The answer at this stage is probably “no”. At present, there is a simple biochemical difference to distinguish C. neoformans and C. gattii with the use of a canavanine-bromothymol-glycine (CBG) agar plate assay that allows separation of the two species by the color of colonies. The species can also be distinguished by an array of sequencing techniques and even MALDI-TOF can rapidly separate species (Posteraro et al., 2012). While these techniques can separate species and genotypes, the ability for the clinician to use this information does not necessarily translate into differences in treatment for C. neoformans vs C. gattii or specific genotypes. There have been clinical comparative analyses between the two species (Smith et al., 2014) but it remains difficult to determine whether differences in therapeutic outcome are due to species variations as opposed to the impact of underlying host immunity.

Epidemiology: The Three-part Outbreak of Cryptococcosis

The understanding of the epidemiology of this disease has been helpful to put the diagnostic strategies in perspective. The appearance of cryptococcosis continues to evolve around the world. It had been estimated that there were greater than 600,000 deaths from cryptococcosis at the peak of the HIV pandemic (Park et al., 2009), but this figure has now been reduced by the impact of effective antiretroviral therapies reaching many locations in the world. Although antiretroviral therapies have not ended the substantial worldwide impact of cryptococcosis in AIDS patients, they have made an impact on its appearance. The second recent clinical cryptococcal outbreak has occurred over the last two decades with C. gattii infections identified in Vancouver, British Columbia and the Northwest USA. The origins of this outbreak have been and continue to be extensively pursued to understand the reason(s) for the appearance of these hypervirulent cryptococcal strains (Billmyre et al., 2014; Engelthaler et al., 2014; Hagen et al., 2013). At least one hypothesis is that climatic changes allowed these cryptococcal strains to compete in the environmental microbiome and with increased exposure infect susceptible hosts. Clinicians need to be aware of this new geographical range of C. gattii in their immunocompetent and immunocompromised populations. There may presently be a reduced number of cases in this particular epidemic in the Pacific Northwest but the range of C. gattii strains appears to be expanding and cases are now being identified in Europe and the eastern USA (Harris et al., 2013).

The other ongoing outbreak of cryptococcosis started in the 1960’s – 1970’s as clinicians developed medical programs in organ transplantation and aggressive treatments of cancers and connective tissue diseases (Pyrgos et al., 2013). With an immunosuppressive armentarium of therapeutics, there has been a steady stream of cryptococcosis cases and with the use of new immunosuppressive biological agents such as anti-TNF and anti-CD54 monoclonal antibodies, the clinician must constantly be aware of cryptococcosis in their differential diagnosis, particularly in high endemic areas and with identified risk factors. For instance, cryptococcosis is widespread in the world but certain areas such as sub-Saharan Africa and the southeastern USA appear to have particularly high (endemic) infection rates. In fact, patients who do not have an underlying HIV infection or are not transplant recipients are remarkably now the highest risk group for mortality in resource-available countries (Bratton et al., 2013). This group, in which mortality can reach >30% in these developed countries, tends to have a delayed diagnosis compared to the HIV and transplant groups and this finding emphasizes that a critical factor in improving outcome is earlier diagnosis of the disease. It is also important to note that this is a heterogeneous group with many underlying diseases that influence outcome; however, there is a subgroup of cases that has no apparent underlying disease yet develops disseminated cryptococcosis. This subgroup generally has a better prognosis for positive outcome and although individuals probably have an altered immune system, it is not as severe as many other patient groups with cryptococcosis. Recently, some of these apparently normal hosts with disseminated cryptococcosis were found to possess an immunological perturbation by producing abundant anti-GM-CSF antibodies (Rosen et al., 2013; Saijo et al., 2014). Furthermore, studies are beginning to identify human genetic variants in immune genes linked to susceptibility for cryptococcosis (Rohatgi et al., 2013). Therefore, risk factors for cryptococcosis are many and can be complex. We can identify some factors clinically but others are buried in our genomes. Thus, the clinician must consider this invasive fungal infection in a variety of circumstances and institute the excellent diagnostic strategies that have been developed.

Diagnosis: The Maturation of Identification

The diagnosis of cryptococcosis, after 100 years of experience, is relatively facile with multiple methods and improved diagnostic strategies. The techniques include direct examination of the fungus in body fluids with India ink examination, histopathology of infected tissue with specific stains to identify capsule (mucicarmine and alcian blue) or presence of melanin (Fontana-Masson), serology from body fluids and culture of fluids and/or tissues. Despite these multiple strategies to make the diagnosis, the clinician, without risk factor assessments and with its non-specific symptoms of the disease, can fail to make an early diagnosis of disseminated cryptococcosis that might be critical to outcome. For example, it has been shown that from the beginning time of symptoms both HIV-infected and transplant patients are diagnosed with cryptococcal meningitis within 2–3 weeks. On the other hand, those without these risk factors may take twice as long to make the diagnosis and patient outcome with treatment is worse (Bratton et al., 2012). Therefore, despite our diagnostic tools, the critical factor for best outcome will be the clinician’s risk assessment and consideration of cryptococcal meningitis in the diagnosis during early symptoms to then apply these diagnostic strategies.

The diagnostic methods such as histopathology and cultures are well-described in a series of reviews and books (Heitman et al., 2011; Perfect, 2013). The diagnostic area with the most recent evolution has been the serological diagnosis of cryptococcosis. The diagnostic use for detection of cryptococcal capsular polysaccharide in serum and cerebrospinal fluid by Latex Agglutination or ELISA has been available for over 35 years and this testing system has an overall sensitivity and specificity of 93–100% and 93–98%, respectively. The false positive rate is less than 1% and generally is explained by technical issues or other infections (including a cross reaction with antigens from Trichosporon species). These tests can occasionally yield a false negative output in early infections, but more commonly can be positive before the detection of viable cryptococcal colonies obtained through cultures.

A lateral flow assay (LFA) was recently introduced into the diagnostic repertoire for cryptococcosis (Hansen et al., 2013; Rugemalila et al., 2013). The semi-quantitative LFA offers many advantages including rapid turnaround time, minimal requirements for a specialized laboratory with potential use at “point of care” and low costs. The LFA has been compared to Latex Agglutination, ELISA and cultures with excellent concordance. It works well in resource-limited settings and can even pick up some C. gattii infections not detected by the other serological tests. This LFA is now being instituted in clinical practice and studies have begun as a “point-of-care” testing for pre-emptive administration of antifungals in resource-limited settings with a high incidence of HIV and cryptococcal diseases. The present hypothesis is that persons with HIV infection and isolated cryptococcal polysaccharide antigenemia benefit from early antifungal therapy to prevent or delay the development of disseminated disease, notably meningitis. For example, prevalence of asymptomatic cryptococcal antigenemia in patients with CD4 cell counts < 100/μl ranges from 2–21% in certain geographical locations (Meya et al., 2010). With an accurate “point of care” test, strategies of diagnostic intervention and pre-emptive therapy are being implemented and tested in Uganda, Rwanda, Mozambique and South Africa. Even in the USA, a recent Centers for Disease Control study found a 3% prevalence in almost 2000 blood samples from HIV-infected patients taken between 1986–2012, a figure that is above the cost-effectiveness threshold for the introduction of a “screen-and-treat” program to effectively manage cryptococcosis during HIV infection (McKenney et al., 2014; Meya et al., 2010). With an accurate “point-of-care” test, these early, precise strategies of diagnostic intervention and pre-emptive therapy should be considered, implemented and/or tested.

Polysaccharide antigen testing has two other important principles. First, a baseline high titer of polysaccharide antigen in serum or CSF carries prognostic significance, in that a high titer (>1:1024) is associated with a large burden of yeasts and a high viable quantitative yeast count in CSF is a predictor of death during systemic antifungal therapy (Jarvis et al., 2014). Second, the elimination kinetics of polysaccharide in the host are not precise and it is important to recognize that the use of changing polysaccharide antigen titers to make therapeutic decisions should be done with caution and in relationship to other clinical factors. Another test in cryptococcal meningitis is the viable quantitative CSF yeast count, that may allow both appreciation of fungal burden and even be used for therapeutic monitoring by measurement of the early fungicidal activity (EFA) with serial quantitative CSF yeast measurements during antifungal treatment (Day et al., 2013). This test has been used as an effective research tool but has not yet been integrated into routine clinical practice. As we consider more useful and precise follow-up lumbar punctures, this measurement could be insightful for management. Furthermore, the ongoing evaluation of EFA as a potential surrogate marker in cryptococcal meningitis is important if it is to be used in the process of regulatory approval of novel anti-cryptococcal agents.

The Evolution of Therapeutic Strategies

The antifungal arsenal for treatment against cryptococcosis currently is largely limited to three old and off-patent drugs, used singly or in combination: amphotericin B (and its liposomal derivatives), 5-fluorocytosine (5FC) and fluconazole. For cryptococcal meningitis, treatment is split into three phases: an initial 2-week induction therapy with a fungicidal amphotericin B-based regimen, followed by 8-week consolidation therapy and subsequently maintenance therapy with fluconazole, continued for 6–12 months and/or until restoration of host immunity (Perfect et al., 2010).

The evidence base for therapy of cryptococcal meningitis, both in the pre- and post-HIV era, is some of the best in all of medical mycology. Numerous trials using mycological endpoints, including time to and rate of CSF sterilization (EFA), demonstrated the superior efficacy of the combination of amphotericin B deoxycholate and 5FC (Bennett et al 1979; Brouwer et al., 2004; Day et al., 2013; Larsen et al., 1990; van der Horst et al., 1997). The place of this combination as the “gold standard” treatment for cryptococcal meningitis is in both the Infectious Disease Society of America and World Health Organization (WHO) Treatment Guidelines and was recently re-affirmed by a large clinical trial in Vietnam (n = 299), showing both a more rapid fungicidal response and a concomitant significant survival advantage for this combination compared to amphotericin B alone (Day et al., 2013).

Despite consistent treatment guideline recommendations from several sources, worldwide access to the antifungal drugs remains woefully inadequate. For example, the main stay fungicidal drug, amphotericin B deoxycholate, use is hampered by cost, inadequate supply chains and difficulties with monitoring and managing its life-threatening adverse events of nephrotoxicity and anaemia (Loyse et al., 2013). Pre-emptive hydration and electrolyte replacement can mitigate some of these side effects and are now recommended by the WHO (Anonymous, 2011). Furthermore, shorter courses of 5–7 days of amphotericin B deoxycholate offer a compromise, with animal model and human data demonstrating similar rates of fungal clearance from the central nervous system in the second week following treatment that is compatible with the drug’s long half-life (Jackson et al., 2012; Livermore et al., 2013; Muzoora et al., 2012). The much costlier liposomal amphotericin B preparations have similar efficacy with fewer toxicities (Hamill et al., 2010) and are widely used in resource-rich settings, particularly for non-HIV patients who are frequently on concomitant nephrotoxic agents and in many clinical situations, such as transplant recipients, these formulations are used as primary therapy. Even in resource-limited areas, a trial exploring the EFA of one to three intermittent high doses (5–10 mg/kg/d) of AmBisome (Gilead Sciences Inc.) is commencing to optimize its safety and long half-life activity in resource-poor settings (AmBition-CM, www.controlled-trials.com/ISRCTN10248064).

Cost and availability are also a challenge with 5FC, as well as adherence to four times daily dosing and bone marrow toxicity. 5FC has just two manufacturers and is not registered in any African country (Loyse et al., 2013). Advocacy efforts are underway to promote generic manufacture of 5FC and encourage the development of a slow release formulation. Fluconazole, used in the consolidation and maintenance phases of treatment as well as in pre-emptive treatment of antigenemic patients as part of pre-ART screening programs, remains widely available via Pfizer’s Diflucan donation program as well as through cheap generics (Loyse et al., 2013). Used for induction therapy, it is fungistatic at the historically-used doses of 400 mg/d (Bicanic et al., 2007). Higher dosing at 800–1200 mg/d (Longley et al., 2008; Mayanja-Kizza et al., 1998), in particular when combined with 5FC (Nussbaum et al., 2010), have improved mycological efficacy and hold promise as an all-oral induction treatment regimen for settings where intravenous amphotericin B simply cannot safely be given. An ongoing ACTG trial of fluconazole is assessing 10-week CSF culture conversion, survival and safety of even higher fluconazole doses of up to 2 g/d [clinicaltrials.gov/show/NCT00885703]. A large multi-site African randomized trial (ACTA, www.controlled-trials.com/ISRCTN45035509) is comparing fluconazole at 1200 mg/d plus 5FC to one or 2-week amphotericin-based induction regimens.

Even with access to amphotericin B based treatment and antiretroviral therapy (ART), early (3-month) mortality outside of clinical trial settings remains high at 35–40% in both some resource-rich and resource-poor settings (Brizendine et al., 2013; Lightowler et al., 2010; Siddiqi et al., 2014). In resource-rich settings, non-HIV, non-transplant patients tend to fare worse, partly because of delayed diagnosis due to lack of clinical suspicion and heterogeneous underlying diseases (Bratton et al., 2013). In HIV-infected patients in resource-poor settings, late presentation is common due to lack of access to diagnostic facilities, which the “point-of-care test” LFA, that also detects cryptococcal antigen in urine, attempts to address. Altered mental status (Glasgow Coma Scale (GCS) < 15 and/or seizures and abnormal behavior) is the most significant predictor of early death (Jarvis et al., 2014) yet its pathophysiology remains poorly understood; we do not even understand whether this is driven by either fungal or host factors.

Raised intracranial pressure is an extremely common complication of cryptococcal meningitis and is associated with sometimes profound and irreversible visual and hearing loss (Graybill et al., 2000). Whilst host factors may play a role, there is evidence for pathophysiology due to fungal factors, with a combination of a high fungal burden and infection with highly encapsulated strains obstructing CSF outflow and causing a communicating hydrocephalus (Bicanic et al., 2009; Robertson et al., 2014). Raised intracranial pressure will resolve with time, so to avert irreversible morbidity, management is by frequent, sometimes daily, large volume (up to 30–40 ml) therapeutic CSF taps or temporary diversion using lumbar or ventricular drains (Perfect et al., 2010). Performance of these interventions is frequently suboptimal in all settings due to doctors’ lack of knowledge, motivation and equipment (disposable manometers) and patients’ fear of lumbar punctures, particularly prevalent in some African countries, but attention to serial lumbar punctures generally has a positive impact on outcome (Rolfes et al., 2014).

The clinical management of disseminated cryptococcosis must critically take into account the infected host’s immune status. Although the disease affects mainly the immunosuppressed host either in HIV-infected or non-HIV-infected (largely iatrogenic immunosuppression for organ transplant or chronic inflammatory conditions) patients, it can also occur in the immunocompetent host, particularly in China and Australia (Chen et al., 2013; Zhu et al., 2010). As a general rule, the ART-naive HIV patient has a high fungal burden with a paucity of inflammation, whilst non-HIV patients and HIV patients presenting post-ART have lower fungal burdens with more CSF inflammation (Bicanic et al., 2007; Bratton et al., 2013). Immunopathology can also vary over time within the same host following restoration of immune function. Corticosteroids that represent a major risk factor for disseminated cryptococcosis can also be needed to control excess inflammation in the treatment of immunocompetent patients with C. gattii infection (Seaton et al., 1997) and for immune reconstitution inflammatory syndrome (IRIS) in HIV and other conditions such as transplant recipients. A large trial is currently addressing the impact of adjunctive dexamethasone on 10-week survival in ART-naive and ART-experienced HIV-infected patients presenting with cryptococcal meningitis (CRYPTODEX, www.controlled-trials.com/ISRCTN59144167).

However, in immunocompromised hosts, augmentation and restoration of host immunity through reversal of immunosuppression or ART in HIV-infected hosts is necessary to clear infection. Interferon gamma is a key pro-inflammatory cytokine (Jarvis et al., 2013) whose levels correlate with rate of fungal clearance and are of prognostic significance (Jarvis et al., 2014; Siddiqui et al., 2005). Augmentation of the poor pro-inflammatory responses characteristic of patients with advanced HIV using interferon-gamma as adjunctive therapy has led to improved fungal clearance (Jarvis et al., 2012), but this recombinant cytokine still primarily is used in the clinics as adjunctive therapy in those who microbiologically fail antifungal therapy.

In HIV patients in whom cryptococcal meningitis usually presents at CD4 counts below 100 cells/μL, ART should be commenced following the completion of the antifungal induction treatment, ideally once CSF is sterile. Competing risks must be balanced: starting ART too early runs the risk of paradoxical IRIS in response to residual fungus and its polysaccharide antigen; starting too late risk patients’ death from other incident opportunistic infections or disease complications. IRIS is characterized by rapid but dysregulated pathogen-specific immune restoration, and can occur in both the HIV and non-HIV-infected host, in response to lowering of doses of immunosuppression (Gupta and Singh, 2011). There are no randomized trial data to inform our management of IRIS: a combination of ART continuation, management of raised intracranial pressure and a short course of tapering doses of steroids for those with persistent symptoms is recommended (Perfect et al., 2010).

Cryptococcal IRIS in the confined space of the central nervous system can be particularly detrimental, occurring in up to 13–30% of HIV-infected patients in published series, usually in the early months on ART (Longley et al., 2013). The recently published COAT trial (n=177) randomized patients to early (1–2 weeks, median 9 days from start of cryptococcal meningitis induction treatment) vs delayed start of ART (5 weeks) (Boulware et al., 2014). There was excess mortality in the early group and thus the trial was stopped early. Although there were no significant differences in IRIS incidence, the difference in mortality appeared to be driven by patients with a lack of CSF inflammation (WBC < 5/μL) at randomization, who died soon after starting ART, with the excess of deaths judged to be due to cryptococcal meningitis. The findings of this trial suggest starting ART between 1 and 2 weeks of induction therapy is too early, but does not inform us about starting ART at later time points between 2 and 5 weeks. Given that high fungal burden and a poor CSF pro-inflammatory response (together with high chemokine levels) pre-ART have both been shown to be risk factors for IRIS (Boulware et al., 2010; Jarvis et al., 2014), a practical point may to use a combination of CSF quantitative cryptococcal cultures and CSF white cell counts at the end of week 2 to determine those patients (sterile CSF with evidence of CSF inflammation) in whom it may be safe to start ART at 3–4 weeks, but this strategy has not been tested.

IRIS can also take the form of “unmasking” IRIS, in patients with previously subclinical infection presenting with cryptococcal meningitis in the first months following start of ART. In African cohorts, 20–30% of new cryptococcal meningitis cases now present in ART-experienced patients. These patients may now be identified and cases prevented with pre-ART screening and treatment of asymptomatic cryptococcal antigenemia with high dose fluconazole (Jarvis et al., 2012). Symptoms as well as the titer of antigenemia may again have prognostic implications and indicate which patients might need referral for a lumbar puncture to rule out meningitis (Jarvis et al., 2009).

In sum, cryptococcosis is in a state of evolution, from the organism, to the host, to the guidelines for diagnosis and treatment. We know a lot but still not enough! The sugar-coated yeast still sickens and we need to manage it better.

Highlights.

  • Understanding cryptococcal epidemiology

  • Molecular pathogenesis and understanding the plasticity in the cryptococcal genome

  • Introduction and integration of new lateral flow antigen test for the diagnosis of cryptococcosis

  • Up-to-date principles of management and anticipated outcome for cryptococcal meningitis.

Footnotes

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Contributor Information

John R Perfect, Division of Infectious Diseases, Department of Medicine, Duke University Medical Center.

Tihana Bicanic, Institute of Infection and Immunity, St. George’s, University of London, London, UK.

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